Background Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with frequent occurrence of epilepsy, autism spectrum disorder (ASD), intellectual disability (ID), and tumors in multiple organs. The aberrant activation of mTORC1 in TSC has led to treatment with mTORC1 inhibitor rapamycin as a lifelong therapy for tumors, but TSC-associated neurocognitive manifestations remain unaffected by rapamycin. Methods Here, we generated patient-specific, induced pluripotent stem cells (iPSCs) from a TSC patient with a heterozygous, germline, nonsense mutation in exon 15 of TSC1 and established an isogenic set of heterozygous (Het), null and corrected wildtype (Corr-WT) iPSCs using CRISPR/Cas9-mediated gene editing. We differentiated these iPSCs into neural progenitor cells (NPCs) and examined neurodevelopmental phenotypes, signaling and changes in gene expression by RNA-seq. Results Differentiated NPCs revealed enlarged cell size in TSC1-Het and Null NPCs, consistent with mTORC1 activation. TSC1-Het and Null NPCs also revealed enhanced proliferation and altered neurite outgrowth in a genotype-dependent manner, which was not reversed by rapamycin. Transcriptome analyses of TSC1-NPCs revealed differentially expressed genes that display a genotype-dependent linear response, i.e., genes upregulated/downregulated in Het were further increased/decreased in Null. In particular, genes linked to ASD, epilepsy, and ID were significantly upregulated or downregulated warranting further investigation. In TSC1-Het and Null NPCs, we also observed basal activation of ERK1/2, which was further activated upon rapamycin treatment. Rapamycin also increased MNK1/2-eIF4E signaling in TSC1-deficient NPCs. Conclusion MEK-ERK and MNK-eIF4E pathways regulate protein translation, and our results suggest that aberrant translation distinct in TSC1/2-deficient NPCs could play a role in neurodevelopmental defects. Our data showing upregulation of these signaling pathways by rapamycin support a strategy to combine a MEK or a MNK inhibitor with rapamycin that may be superior for TSC-associated CNS defects. Importantly, our generation of isogenic sets of NPCs from TSC patients provides a valuable platform for translatome and large-scale drug screening studies. Overall, our studies further support the notion that early developmental events such as NPC proliferation and initial process formation, such as neurite number and length that occur prior to neuronal differentiation, represent primary events in neurogenesis critical to disease pathogenesis of neurodevelopmental disorders such as ASD.

中文翻译：

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TSC患者来源的等基因神经祖细胞揭示了改变的早期神经发育表型和雷帕霉素诱导的MNK-eIF4E信号传导。

背景结节性硬化症（TSC）是一种神经发育障碍，经常发生癫痫，自闭症谱系障碍（ASD），智障（ID）和多器官肿瘤。TSC中mTORC1的异常激活已导致使用mTORC1抑制剂雷帕霉素治疗肿瘤的终生疗法，但与TSC相关的神经认知表现仍不受雷帕霉素影响。方法在这里，我们从TSC患者的TSC1外显子15杂合，种系，无义突变中产生了患者特异性的诱导多能干细胞（iPSC），并建立了一个同基因组杂合（Het），无效和校正野生型（Corr -WT）使用CRISPR / Cas9介导的基因编辑的iPSC。我们将这些iPSC分化为神经祖细胞（NPC），并检查了神经发育表型，信号和RNA表达的基因表达变化。结果分化的NPC显示TSC1-Het和Null NPC中细胞大小增大，与mTORC1激活一致。TSC1-Het和Null NPC也以基因型依赖的方式显示出增强的增殖和改变的神经突生长，雷帕霉素并不能逆转。TSC1-NPC的转录组分析显示差异表达的基因显示出基因型依赖性线性反应，即在Het中上调/下调的基因在Null中进一步增加/减少。特别是，与ASD，癫痫和ID相关的基因显着上调或下调，有待进一步研究。在TSC1-Het和Null NPC中，我们还观察到ERK1 / 2的基础活化，该活化在雷帕霉素治疗后进一步活化。雷帕霉素还在缺乏TSC1的NPC中增加MNK1 / 2-eIF4E信号传导。结论MEK-ERK和MNK-eIF4E途径调节蛋白质翻译，我们的结果表明，在TSC1 / 2缺失的NPC中异常翻译异常可能在神经发育缺陷中起作用。我们的数据显示雷帕霉素对这些信号通路的上调支持了将MEK或MNK抑制剂与雷帕霉素结合的策略，这种策略可能优于TSC相关的CNS缺陷。重要的是，我们从TSC患者中产生的等基因NPC集为转位组和大规模药物筛选研究提供了宝贵的平台。总体而言，我们的研究进一步支持以下观点：早期发育事件，例如NPC增殖和初始过程形成，例如神经元分化之前发生的神经突数量和长度，